Nucleic acid molecules encoding gpr84

ABSTRACT

The present invention is an isolated nucleic acid molecule encoding CD36, G qi9 , and G protein-coupled receptor 84 (GPR84) proteins as well as vectors and recombinant host cells which co-express CD36, G qi9 , and GPR84 proteins for use in identifying modulators of GPR84 activity.

BACKGROUND OF THE INVENTION

Taste signaling is found throughout the animal kingdom, from simplemetazoans to the most complex of vertebrates. Taste sensation isbelieved to be mediated by receptors, i.e., metabotropic or inotropicreceptors. Cells which express taste receptors, when exposed to certainchemical stimuli, elicit taste sensation by depolarizing to generate anaction potential, which triggers the sensation. As such, taste receptorsspecifically recognize molecules that elicit specific taste sensation.These molecules are also referred to herein as “tastants.” Many tastereceptors belong to the 7-transmembrane receptor superfamily (Hoon, etal. (1999) Cell 96:451; Adler, et al. (2000) Cell 100:693), which arealso known as G protein-coupled receptors (GPCRs). Other tastes arebelieved to be mediated by channel proteins.

The biochemical analysis and molecular cloning of a number of suchreceptors has revealed basic principles regarding the function of thesereceptors. For example, U.S. Pat. No. 5,691,188 describes how upon aligand binding to a GPCR, the receptor presumably undergoes aconformational change leading to activation of the G protein. G proteinsare comprised of three subunits: a guanyl nucleotide binding α subunit,a β subunit, and a γ subunit. When GDP is bound, the G protein exists asa heterotrimer, the Gαβγ complex. When GTP is bound, the α subunitdissociates from the heterotrimer, leaving a Gβγ complex. When a Gαβγcomplex operatively associates with an activated G protein-coupledreceptor in a cell membrane, the rate of exchange of GTP for bound GDPis increased and the rate of dissociation of the bound Gα subunit fromthe Gαβγ complex increases. The free Gα subunit and Gβγ complex are thuscapable of transmitting a signal to downstream elements of a variety ofsignal transduction pathways. These events form the basis for amultiplicity of different cell signaling phenomena, including forexample the signaling phenomena that are identified as neurologicalsensory perceptions such as taste and/or smell.

Mammals are believed to have five basic taste modalities, sweet, bitter,sour, salty, and umami (the taste of monosodium glutamate). See, e.g.,Kinnamon, et al. (1992) Ann. Rev. Physiol. 54:715-31; Lindemann (1996)Physiol. Rev. 76:718-66; Stewart, et al. (1997) Am. J Physiol. 272:1-26.Numerous physiological studies in animals have shown that taste receptorcells may selectively respond to different chemical stimuli. See, e.g.,Akabas, et al. (1988) Science 242:1047-50; Gilbertson, et al. (1992) J.Gen. Physiol. 100:803-24; Bernhardt, et al. (1996) J. Physiol.490:325-36; Cummings, et al. (1996) J. Neurophysiol. 75:1256-63.

Much is known about the psychophysics and physiology of taste cellfunction. Thus, the identification and isolation of novel tastereceptors and taste signaling molecules could allow for new methods ofchemical and genetic modulation of taste transduction pathways. Forexample, the availability of receptor and channel molecules permits thescreening for high affinity agonists, antagonists, inverse agonists, andmodulators of taste activity. Such taste modulating compounds could beuseful in the pharmaceutical and food industries to improve the taste ofa variety of consumer products, or to block undesirable tastes, e.g., incertain pharmaceuticals.

Complete or partial sequences of various human and other eukaryoticchemosensory receptors are known. See, e.g., Pilpel & Lancet (1999)Protein Science 8:969-977; Mombaerts (1999) Annu. Rev. Neurosci.22:487-50; Wang, et al. (2006) J. Biol. Chem. M608019200. See also, U.S.Pat. No. 5,874,243, WO 92/17585, WO 95/18140, WO 97/17444, WO 99/67282,and WO 2007/027661. In addition, the identification of ligands for Gprotein coupled receptors such as GPR84 has been conducted. See Wang, etal. (2006) supra; WO 2007/027661; and U.S. Patent Application No.2007/0196867.

SUMMARY OF THE INVENTION

The present invention is an isolated nucleic acid molecule encodingCD36, G_(qi9), and G protein-coupled receptor 84 (GPR84) proteins.Expression vectors with inducible or constitutive expression of CD36,G_(qi)9, and GPR84 are also embraced by the present invention, as is anisolated recombinant host cell which co-expresses CD36, G_(qi9), andGPR84 proteins.

The present invention is also a method for identifying a modulator ofGPR84 activity. The method involves contacting a recombinant host cell,which co-expresses CD36, G_(qi9), and GPR84 proteins, with a testcompound and determining the functional effect of the test compound onGPR84 activity thereby identifying a modulator of GPR84.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides isolated nucleic acid molecules encodingG protein-coupled receptor 84 (GPR84), CD36 and G_(qi9), and use of thesame in the identification of ligands for GPR84, e.g., agonists andantagonists. In particular, the present invention relates to GPR84 as afat taste receptor and use of the same in screening assays for compoundsthat mimic fat taste.

As used herein, the term “isolated,” when referring to a nucleic acidmolecule refers to a state of purification or concentration differentthan that which occurs naturally. The nucleic acid molecules describedherein may be isolated or otherwise associated with structures orcompounds to which they are not normally associated in nature, accordingto a variety of methods and processed known to those of skill in theart.

GPR84 has been identified as a receptor for medium-chain free fattyacids (Wang, et al. (2006) supra). The GPR84 protein is found in avariety of species including human, mouse, rat, and the like and nucleicmolecules encoding any one of these proteins can be used in accordancewith the present invention. In this regard, the present inventionembraces nucleic acid molecules encoding GPR84 proteins listed in Table1.

TABLE 1 GENBANK GENBANK Accession No. SEQ Accession No. SEQ % of AminoAcid ID of Nucleic Acid ID Iden- Source Sequence NO: Sequence NO: tity*Homo NP_065103 1 NM_020370 2  100% sapiens Mus NP_109645 3 NM_030720 485.4% musculus Rattus NP_001102979 5 NM_001109509 6 83.8% norvegicus Bostaurus NP_001033657 7 NM_001038568 8 89.6% *% Identity based onalignment with human GPR84 protein.

In some embodiments, the nucleic acid molecule encodes a human GPR84protein. In other embodiments, the nucleic acid molecule encodes a humanGPR84 protein of SEQ ID NO:1. In particular embodiments, the nucleicacid molecule encoding human GRP84 protein is set forth in SEQ ID NO:9.

Functionally, the GPR84 proteins disclosed herein are members of afamily of related seven transmembrane G protein-coupled receptors, whichare believed to be involved in taste transduction. Structurally, thenucleotide sequences of the GPR84 family members can encode relatedproteins including an extracellular domain, seven transmembrane domains,and a cytoplasmic domain. Related GPR84 family members from otherspecies share at least about 60%, 70%, 80%, or 90% protein sequenceidentity over a region of at least about 50 amino acid residues inlength, optionally 100, 200, 300, or more amino acid residues in lengthto SEQ ID NOs: 1, 3, 5 or 7.

Specific regions of the GPR84 amino acid sequences can be used toidentify polymorphic variants, interspecies homologs, and alleles ofGPR84 family members. This identification can be made in vitro, e.g.,under stringent hybridization conditions or PCR (e.g., using primersencoding GPR84 consensus sequences), or by using the sequenceinformation in a computer system for comparison with other nucleotidesequences. Different alleles of GPR84 genes within a single speciespopulation will also be useful in determining whether differences inallelic sequences correlate to differences in taste perception betweenmembers of the population. Classical PCR-type amplification and cloningtechniques are useful for isolating orthologs, for example, wheredegenerate primers are sufficient for detecting related genes acrossspecies, which typically have a higher level of relative identity thanparalogous members of the GPR84 family within a single species.

Typically, identification of polymorphic variants and alleles of GPR84family members can be made by comparing an amino acid sequence of about25 amino acids or more, e.g., 50-100 amino acids. Amino acid identity ofapproximately at least 60%, 70%, 75%, 80%, 85%, 90%, 95-99%, or abovetypically demonstrates that a protein is a polymorphic variant,interspecies homolog, or allele of a T1R family member. Sequencecomparison can be performed using any conventional sequence comparisonalgorithms. Antibodies that bind specifically to GPR84 polypeptides or aconserved region thereof can also be used to identify alleles,interspecies homologs, and polymorphic variants.

In addition to GRP84, the nucleic acid molecule of the invention furtherencodes a chimeric guanine nucleotide-binding protein (G protein)composed of the G protein α_(q) subunit, wherein the C-terminal nineresidues have been replaced by those of the G protein α_(i) subunit.This chimeric G protein, termed G_(qi9), is known in the art anddescribed by Parmentier, et al. (1998) Mol. Pharmacol. 53(4):778-86;Perroy, et al. ((2001) J. Biol. Chem. 276: 45800-45805) for couplingreceptors to phospholipase C. By way of illustration, the nineC-terminal amino acid residues of the G_(q) subunit set forth in GENBANKAccession No. NP_(—)002063.2 (SEQ ID NO:10) are replaced with the nineC-terminal amino acid residues of the G protein α_(i) subunit, i.e.,Asn-Asn-Leu-Lys-Asp-Cys-Gly-Leu-Phe (SEQ ID NO:11). While the humanG_(q) subunit is desirably employed in the present invention, non-humanG_(q) subunit can also be used. For example, the G_(q) subunits listedin Table 2 would be suitable for use in the present invention.

TABLE 2 GENBANK GENBANK Accession No. SEQ Accession No. SEQ of AminoAcid ID of Nucleic Acid ID % Source Sequence NO: Sequence NO: Identity*Homo NP_002063 10 NM_002072 12  100% sapiens Mus NP_032165 13 NM_00813914 99.7% musculus Rattus NP_112298 15 NM_031036 16 99.4% norvegicus *%Identity based on alignment with human G_(q) protein subunit.

G_(q) subunits of use in accordance with the present invention desirablyshare at least about 90% protein sequence identity over a region of atleast about 50 amino acid residues in length, optionally 100, 200, 300,or more amino acid residues in length to SEQ ID NOs: 10, 13, or 15. Asdescribed for GPR84, related sequences can be identified using a varietyof conventional methods, using stringent hybridization conditions orantibody cross-reactivity.

Further included in the nucleic acid molecule of the present inventionare sequences encoding CD36. It has been suggested that GPR120 and CD36form an independent pathway for fatty acid reception (Matsumura, et al.(2007) Biomed. Res. 28:49-55). Moreover, it is contemplated that CD36also functions in conjunction with GPR84 in fatty acid transport andsensing of dietary lipids. Thus, the present invention embraces theco-expression of CD36 with GPR84 and G_(qi9). While the human CD36 isdesirably employed in the present invention, non-human CD36 can also beused. For example, the CD36 proteins listed in Table 3 would be suitablefor use in the present invention.

TABLE 3 GENBANK GENBANK Accession No. SEQ Accession No. SEQ % of AminoAcid ID of Nucleic Acid ID Iden- Source Sequence NO: Sequence NO: tity*Homo NP_001001547 17 NM_001001547 18  100% sapiens Mus NP_031669 19NM_007643 20 86.4% musculus Rattus NP_113749 21 NM_031561 22 87.9%norvegicus *% Identity based on alignment with human CD36 protein.

In so far as the CD36 proteins disclosed herein function as fatty acidtransporters, the present invention embraces other CD36 proteins sharingat least about 90% protein sequence identity over a region of at leastabout 50 amino acid residues in length, optionally 100, 200, 300, ormore amino acid residues in length to SEQ ID NOs: 17, 19, or 21.

The amino acid sequences of the proteins of the invention can beidentified by putative translation of the coding nucleic acid sequences.These various amino acid sequences and the coding nucleic acid sequencescan be compared to one another or to other sequences according to anumber of methods.

For example, in sequence comparison, typically one sequence acts as areference sequence, to which test sequences are compared. When using asequence comparison algorithm, test and reference sequences are enteredinto a computer, subsequence coordinates are designated, if necessary,and sequence algorithm program parameters are designated. Defaultprogram parameters can be used, as described below for the BLASTN andBLASTP programs, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window,” as used herein, includes reference to a segmentof any one of the number of contiguous positions, e.g., from 20 to 600,usually about 50 to about 200 or about 100 to about 150 in which asequence can be compared to a reference sequence of the same number ofcontiguous positions after the two sequences are optimally aligned.Methods of alignment of sequences for comparison are well-known in theart. Optimal alignment of sequences for comparison can be conducted,e.g., by the local homology algorithm of Smith & Waterman ((1981) Adv.Appl. Math. 2:482), by the homology alignment algorithm of Needleman &Wunsch ((1970) J. Mol. Biol. 48:443), by the search for similaritymethod of Pearson & Lipman ((1988) Proc. Natl. Acad Sci. USA 85:2444),by computerized implementations of these algorithms (GAP, BESTFIT,FASTA, and TFASTA in the Wisconsin Genetics Software Package, GeneticsComputer Group, Madison, Wis.), or by manual alignment and visualinspection (see, e.g., Current Protocols in Molecular Biology (1995)Ausubel, et al., eds. supplement).

A preferred example of an algorithm that is suitable for determiningpercent sequence identity are the BLAST and BLAST 2.0 algorithms, whichare described by Latched, et al. (1977) Nuc. Acids Res. 25:3389-3402,and Altschul, et al. (1990) J. Mol Biol. 2 15:403-410, respectively.Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information. This algorithm involvesfirst identifying high scoring sequence pairs (HSPs) by identifyingshort words of length W in the query sequence, which either match orsatisfy some positive-valued threshold score T when aligned with a wordof the same length in a database sequence. T is referred to as theneighborhood word score threshold (Altschul, et al. (1990) supra;Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T, and Xdetermine the sensitivity and speed of the alignment. The BLASTN program(for nucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) or 10, M=5, N=4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10 and the BLOSUM62 scoring matrix (see,Henikoff & Henikoff (1989) Proc. Nati. Acad. Sci. USA 89:10915)alignments (B) of 50, expectation (E) of 10, M=5, Nz=4, and a comparisonof both strands.

Another example of a useful algorithm is PILEUP. PILEUP creates amultiple sequence alignment from a group of related sequences usingprogressive, pairwise alignments to show relationship and percentsequence identity. It also plots a so-called “tree” or “dendogram”showing the clustering relationships used to create the alignment.PILEUP uses a simplification of the progressive alignment method of Feng& Doolittle ((1987) J. Mol. Evol. 35:351-360). The method used issimilar to the method described by Higgins & Sharp ((1989) CABIOS5:151-153). The program can align up to 300 sequences, each of a maximumlength of 5,000 nucleotides or amino acids. The multiple alignmentprocedure begins with the pairwise alignment of the two most similarsequences, producing a cluster of two aligned sequences. This cluster isthen aligned to the next most related sequence or cluster of alignedsequences. Two clusters of sequences are aligned by a simple extensionof the pairwise alignment of two individual sequences. The finalalignment is achieved by a series of progressive, pairwise alignments.The program is run by designating specific sequences and their aminoacid or nucleotide coordinates for regions of sequence comparison and bydesignating the program parameters. Using PILEUP, a reference sequenceis compared to other test sequences to determine the percent sequenceidentity relationship using the following parameters: default gap weight(3.00), default gap length weight (0.10), and weighted end gaps. PILEUPcan be obtained from the GCG sequence analysis software package(Devereaux, et al. (1984) Nuc. Acids Res. 12:387-395).

As shown herein, comparison of the human protein sequences of theinvention to all known proteins in the public sequence databases usingBLASTP algorithm revealed strong homology to other GPR84, G_(q) subunitsand CD36 proteins having at least about 80% amino acid identity thehuman sequences.

As an alternative to direct amino acid or nucleic acid sequencecomparisons, suitable GPR84, G_(q) subunit and CD36 proteins can beidentified by hybridization of nucleic acid molecules (e.g., the nucleicacid molecules disclosed in Tables 1-3, or probes thereof) understringent hybridization conditions to nucleic acids from other species(e.g., genomic or cDNA sequences). The phrase “stringent hybridizationconditions” refers to conditions under which a probe will hybridize toits target subsequence, typically in a complex mixture of nucleic acid,but to no other sequences. Stringent conditions are sequence-dependentand will be different in different circumstances. Longer sequenceshybridize specifically at higher temperatures. An extensive guide to thehybridization of nucleic acids is found in Tijssen (1993) Techniques inBiochemistry and Molecular Biology, Hybridization with Nucleic Probes,“Overview of principles of hybridization and the strategy of nucleicacid assays”. Generally, stringent conditions are selected to be about5-10° C. lower than the thermal melting point (T_(m)) for the specificsequence at a defined ionic strength pH. The T_(m) is the temperature(under defined ionic strength, pH, and nucleic concentration) at which50% of the probes complementary to the target hybridize to the targetsequence at equilibrium (as the target sequences are present in excess,at T_(m), 50% of the probes are occupied at equilibrium). Stringentconditions will be those in which the salt concentration is less thanabout 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ionconcentration (or other salts) at pH 7.0 to 8.3 and the temperature isat least about 30° C. for short probes (e.g., 10 to 50 nucleotides) andat least about 60° C. for long probes (e.g., greater than 50nucleotides). Stringent conditions can also be achieved with theaddition of destabilizing agents such as formamide. For selective orspecific hybridization, a positive signal is at least two timesbackground, optionally 10 times background hybridization. Exemplarystringent hybridization conditions can be as follows: 50% formamide,5×SSC, and 1% SDS with incubation at 42° C. (or 5×SSC, 1% SDS withincubation at 65° C.) with a wash in 0.2×SSC and 0.1% SDS at 65° C. Suchhybridizations and wash steps can be carried out for, e.g., 1, 2, 5, 10,15, 30, 60, or more minutes.

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially related if the polypeptides whichthey encode are substantially related. This occurs, for example, when acopy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.Exemplary “moderately stringent hybridization conditions” include ahybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1×SSC at 45° C. Such hybridizations and wash steps can becarried out for, e.g., 1, 2, 5, 10, 15, 30, 60, or more minutes. Apositive hybridization is at least twice background. Those of ordinaryskill will readily recognize that alternative hybridization and washconditions can be utilized to provide conditions of similar stringency.

As is clear from the present disclosure, the nucleic acid moleculesencoding the GPR84, G_(q) subunit and CD36 proteins of the invention canbe isolated from a variety of sources, genetically engineered,amplified, synthesized, and/or expressed recombinantly according tomethods well-known in the art. Isolation and expression of the proteinsof the present invention can be performed as described below. PCRprimers can be used for the amplification of nucleic acids encodingGPR84, G_(q) subunit and CD36 proteins, and libraries of these nucleicacids can optionally be generated and screened for the identification ofa suitable nucleic acid molecule for protein expression. Amplificationmethods are well-known in the art, and include, e.g., polymerase chainreaction, PCR (PCR Protocols, a Guide to Methods and Applications (1990)ed. Innis. Academic Press, New York, and PCR Strategies (1995) ed.Innis, Academic Press, Inc., NY); ligase chain reaction (LCR) (see,e.g., Wu (1989) Genomics 4:560; Landegren (1988) Science 241:1077;Barringer (1990) Gene 89:117); transcription amplification (see, e.g.,Kwoh (1989) Proc. Natl. Acad. Sci. USA 86:1173); self-sustained sequencereplication (see, e.g., Guatelli (1990) Proc. Natl. Acad. Sci. USA87:1874); Q Beta replicase amplification (see, e.g., Smith (1997) J.Clin. Microbiol. 35:1477-1491); automated Q-beta replicase amplificationassay (see, e.g., Burg (1996) Mol. Cell. Probes 10:257-271) and otherRNA polymerase-mediated techniques (e.g., NASBA; Cangene, Mississauga,Ontario). Primers can be designed to retain the original sequence of theprotein of interest. Alternatively, the primers can encode amino acidresidues that are conservative substitutions (e.g., hydrophobic forhydrophobic residue or functionally benign substitutions (e.g., do notprevent plasma membrane insertion, cause cleavage by peptidase, causeabnormal folding of receptor, and the like). Once amplified, the nucleicacids, either individually or as libraries, can be cloned according tomethods known in the art. Indeed, techniques for the manipulation ofnucleic acids, such as, for example, for generating mutations insequences, subcloning, labeling probes, sequencing, hybridization andthe like are well-described in the scientific and Patent literature.See, e.g., Sambrook, ed. (1989) Molecular Cloning: a Laboratory Manual(2nd ed.), Vols. 1-3, Cold Spring Harbor Laboratory, and CurrentProtocols in Molecular Biology (1997) Ausubel, ed. John Wiley & Sons,Inc., New York.

As an alternative to amplification and/or cloning, nucleic acids of theinvention can be synthesized in vitro by well-known chemical synthesistechniques, as described in, e.g., Carruthers (1982) Cold Spring HarborSymp. Quant. Biol. 47:411-418; Adams (1983) Am. Chem. Soc. 105:661;Belousov (1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995) FreeRadic. Biol. Med. 19:373-380; Blommers (1994) Biochemistry 33:7886-7896;Narang (1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68:109.Double-stranded DNA fragments can then be obtained either bysynthesizing the complementary strand and annealing the strands togetherunder appropriate conditions, or by adding the complementary strandusing DNA polymerase with an appropriate primer sequence.

Once obtained, nucleic acids encoding the GPR84, G_(q) subunit and CD36proteins are introduced into an expression vector for recombinantprotein expression. Depending on the intended use (e.g., in phenotypicor ligand binding assays), any expression vector system can be used,including, in addition to mammalian cells, e.g., bacterial, yeast,insect, or plant systems. As used herein, the term “expression vector”refers to any recombinant expression system for the purpose ofexpressing a nucleic acid molecule of the invention, constitutively orinducibly, in any cell, including prokaryotic, yeast, fungal, plant,insect or mammalian cell. The term includes linear or circularexpression systems. The term includes expression systems that remainepisomal or integrate into the host cell genome. The expression systemscan have the ability to self-replicate or not, i.e., drive onlytransient expression in a cell. The term includes recombinant expressioncassettes which contain only the minimum elements needed fortranscription of the recombinant nucleic acid.

To achieve recombinant protein expression, it is desirable that thenucleic acids of the invention are operably linked to transcriptional ortranslational control elements, e.g., transcription and translationinitiation sequences, promoters and enhancers, transcription andtranslation terminators, polyadenylation sequences, and other sequencesuseful for transcribing DNA into RNA. In construction of recombinantnucleic acid molecules and vectors, a promoter can be operably-linked toone or more nucleic acid molecules of the invention to direct expressionof the desired nucleic acid in all or a subset of cells or tissues. A“promoter” is defined as an array of nucleic acids that directtranscription of a nucleic acid. As used herein, a promoter includesnecessary nucleic acid sequences near the start site of transcription,such as, in the case of a polymerase II type promoter, a TATA element. Apromoter also optionally includes distal enhancer or repressor elements,which can be located as much as several thousand base pairs from thestart site of transcription. In particular embodiments, constitutive orinducible promoters are employed. A “constitutive” promoter is apromoter that is active under most environmental and developmentalconditions. Examples of constitutive promoters include, but are notlimited to, the P-actin promoter and the CMV promoter. An “inducible”promoter is a promoter that is active under environmental ordevelopmental regulation. Examples of inducible promoters include, butare not limited to, the human c-fos promoter, steroid-induciblepromoters such as a glucocorticoid-inducible promoter, and smallmolecule inducible promoters such as the tetracycline regulatedpromoter. Promoters of the invention are The term “operably-linked”refers to a functional linkage between a nucleic acid expression controlsequence (such as a promoter, or array of transcription factor bindingsites) and a second nucleic acid molecule, wherein the expressioncontrol sequence directs transcription of the nucleic acid correspondingto the second sequence.

In accordance with some embodiments of the present invention, thenucleic acid molecules encoding GPR84, CD36 and G_(qi9) are introduced,in tandem, into one expression vector for expression as individualproteins in a host cell. In other embodiments, the nucleic acidmolecules encoding GPR84, CD36 and G_(qi9) are each introduced intodifferent expression vectors for co-expression in a host cell. In yetother embodiments, two expression vectors are employed with variouscombinations of nucleic acid molecules encoding GPR84, CD36 and G_(qi9)(e.g., one expression vector harbors GPR84 and G_(qi9) nucleic acids andthe other expression vector harbors CD36 nucleic acids). In addition, itis further contemplated that the proteins of the present invention canbe expressed as fusion proteins, e.g., a GPR84-G_(qi9) fusion protein.

Wherein the nucleic acid molecules encoding GPR84, CD36 and G_(qi9) areintroduced, in tandem, into one expression vector for co-expression ofindividual proteins in a host cell, some embodiments embrace the use ofone promoter for regulating the expression of all three proteins. Inthis regard, it is contemplated that the nucleic acid molecules encodingGPR84, CD36 and G_(qi9) are separated from each other by InternalRibosome Entry Sites (IRESs). IRES elements are known from viral andmammalian genes (Martinez-Salas (1999) Curr. Opin. Biotechnol.10:458-64), and have also been identified in screens of small syntheticoligonucleotides (Venkatesan & Dasgupta (2001) Mol. Cell. Biol.21:2826-37.). The IRES from the encephalomyocarditis virus has beenanalyzed in detail (Mizuguchi, et al. (2000) Mol. Ther. 1:376-82). AnIRES is an element encoded in DNA that results in a structure in thetranscribed RNA at which eukaryotic ribosomes can bind and initiatetranslation. An IRES permits two or more proteins to be produced from asingle RNA molecule (the first protein is translated by ribosomes thatbind the RNA at the cap structure of its 5′ terminus, (Martinez-Salas(1999) supra).

Alternatively, other embodiments embrace tandem introduction of nucleicacid molecules encoding GPR84, CD36 and G_(qi9) into one expressionvector, wherein the coding sequence for each protein has its ownregulatory sequences. In this regard, specific embodiments of theinvention include a nucleic acid molecule containing, in order, thefollowing sequences: promoter¹→CD36 coding sequence→polyAsignal→promoter²→G_(qi9) coding sequence→polyA signal→promoter³→GPR84coding sequence→polyA signal, wherein promoter¹⁻³ can be the same ordifferent, or inducible or constitutive.

Expression vectors, either as individual expression vectors forco-expressing GPR84, CD36 or G_(qi9) proteins, or one expression vectorharboring nucleic acids encoding GPR84, CD36 and G_(qi9) proteins can beintroduced into a genome or into the cytoplasm or a nucleus of a hostcell and expressed by a variety of conventional techniqueswell-described in the scientific and Patent literature. See, e.g.,Roberts, (1987) Nature 328:731; and Sambrook, ed. (1989) supra). Productinformation from manufacturers of biological reagents and experimentalequipment also provide information regarding known biological methods.The vectors can be isolated from natural sources, obtained from suchsources as ATCC or GENBANK libraries, or prepared by synthetic orrecombinant methods.

The nucleic acids of the invention can be expressed in expressioncassettes or vectors (including plasmids and viruses) which are stablyor transiently maintained in cells (e.g., episomal expression systems).Selection markers can be incorporated into expression cassettes andvectors to confer a selectable phenotype on transformed cells andsequences. For example, selection markers can code for episomalmaintenance and replication such that integration into the host genomeis not required. For example, the marker may encode antibioticresistance (e.g., chloramphenicol, kanamycin, G418, bleomycin,hygromycin) or herbicide resistance (e.g., chlorosulfuron) to permitselection of those cells transformed with the desired DNA sequences(see, e.g., Blondelet-Rouault (1997) Gene 190:315-317; Aubrecht (1997)J. Pharmacol. Exp. Ther. 281:992-997).

Also within the scope of the invention are recombinant host cells forco-expressing the proteins of the invention. Proteins of the inventionare said to be “co-expressed” in that the host cell, when transformedwith nucleic acids encoding GPR84, CD36 or G_(qi9) proteins, transcribesand translates GPR84, CD36 or G_(qi9) proteins. By “host cell” is meanta cell that contains an expression vector and supports the replicationor expression of the expression vector. Host cells can be prokaryoticcells such as E. coli, or eukaryotic cells such as yeast, insect,amphibian, or mammalian cells such as CHO, HeLa, HEK-293, and the like,e.g., cultured cells, explants, and cells in vivo.

As indicated, the host cell can be mammalian or non-mammalian, withpreference given to mammalian host cells. Any of the well-knownprocedures for introducing the expression vector(s) of the inventioninto host cells can be used. These include the use of calcium phosphatetransfection, polybrene, protoplast fusion, electroporation, liposomes,microinjection, plasma vectors, viral vectors and any of the otherwell-known methods for introducing cloned genomic DNA, cDNA, syntheticDNA or other foreign genetic material into a host cell (see, e.g.,Sambrook et al. supra) After the expression vector is introduced intothe host cells, the transfected cells are cultured under conditionsfavoring expression of the proteins of the invention. Host cells of theinvention which co-express GPR84, CD36 or G_(qi9) proteins from one ormore expression vectors find application in analyzing fatty acidtransport and sensing of dietary lipids as well as in methods foridentifying agents which modulate GPR84 activity.

Accordingly, the invention also provides methods of screening formodulators, e.g., activators, inhibitors, stimulators, enhancers,agonists, and antagonists, of GPR84 activity. Such modulators of GPR84activity are useful for pharmacological, chemical, and geneticmodulation of taste signaling pathways. These methods of screening canbe used to identify high affinity agonists and antagonists of taste cellactivity. These modulatory compounds can then be used in the food andpharmaceutical industries to customize taste, e.g., to modulate thetastes of foods.

“Inhibitors,” “activators,” and “modulators” of GPR84 activity are usedinterchangeably to refer to inhibitory, activating, or modulatingmolecules identified using assays of the invention. Inhibitors arecompounds that, e.g., bind to, partially or totally block stimulation,decrease, prevent, delay activation, inactivate, desensitize, or downregulate GPR84 activity, e.g., antagonists. Activators are compoundsthat, e.g., bind to, stimulate, increase, open, activate, facilitate,enhance activation, sensitize, or up regulate GPR84 activity, e.g.,agonists. Modulators also include compounds that, e.g., alter theinteraction of a receptor with extracellular proteins that bindactivators or inhibitors, G proteins, or kinases. Such assays forinhibitors and activators include, e.g., expressing GPR84, CD36 andG_(qi9) in cells or cell membranes, applying putative modulatorcompounds and then determining the functional effects on tastetransduction. Results of assays with potential activators, inhibitors,or modulators are compared to control samples without the inhibitor,activator, or modulator to examine the extent of modulation of GPR84activity. Control samples (untreated with modulators) are assigned arelative GPR84 activity value of 100%. Inhibition of a GPR84 is achievedwhen the GPR84 activity value relative to the control is about 80%,optionally 50% or 25-0%. Activation of a GPR84 is achieved when theGPR84 activity value relative to the control is 110%, optionally 150%,optionally 200-500%, or 1000-3000% higher.

Thus, the invention provides assays for detecting and characterizingtaste modulation, wherein GPR84 is used as a reporter molecule fordetermining the functional effect of modulators on fatty acid tastetransduction. The phrase “functional effect(s)” in the context of assaysfor testing compounds that modulate GPR84 activity includes thedetermination of any parameter that is indirectly or directly under theinfluence of the receptor, e.g., functional, physical and chemicaleffects. It includes ligand binding, changes in ion flux, membranepotential, current flow, transcription, G protein binding, GPCRphosphorylation or dephosphorylation, signal transduction,receptor-ligand interactions, second messenger concentrations (e.g.,cAMP, cGMP, IP₃, or intracellular Ca²⁺).

By “determining the functional effect” in the context of assays hereinis meant assays for a compound that increases or decreases a parameterthat is indirectly or directly under the influence of GPR84, e.g.,functional, physical and chemical effects. Such functional effects canbe measured by any means known to those skilled in the art, e.g.,changes in spectroscopic characteristics (e.g., fluorescence,absorbance, refractive index), hydrodynamic (e.g., shape),chromatographic, or solubility properties; patch clamping;voltage-sensitive dyes; whole cell currents; radioisotope efflux;inducible markers; ligand-binding assays; voltage, membrane potentialand conductance changes; ion flux assays; changes in intracellularsecond messengers such as cAMP, cGMP, and inositol triphosphate (IP₃);changes in intracellular calcium levels; and the like.

Taste receptors bind tastants and initiate the transduction of chemicalstimuli into electrical signals. An activated or inhibited G proteinwill in turn alter the properties of target enzymes, channels, and othereffector proteins. Some examples are the activation of cGMPphosphodiesterase by transducin in the visual system, adenylate cyclaseby the stimulatory G protein, phospholipase C by G_(q) and other cognateG proteins, and modulation of diverse channels by G_(i) and other Gproteins. Downstream consequences can also be examined such asgeneration of diacyl glycerol and IP₃ by phospholipase C, and in turn,for calcium mobilization by IP₃. Accordingly, in one embodiment,activation of GPR84 can be detected using monitoring changes inintracellular calcium by detecting FURA-2-dependent fluorescence in thehost cell.

Activated GPCR receptors become substrates for kinases thatphosphorylate the C-terminal tail of the receptor (and possibly othersites as well). Thus, activators can promote the transfer of ³²P fromgamma-labeled GTP to the receptor, which can be assayed with ascintillation counter. The phosphorylation of the C-terminal tail willpromote the binding of arrestin-like proteins and will interfere withthe binding of G proteins. The kinase/arrestin pathway plays a key rolein the desensitization of many GPCR receptors. For example, compoundsthat modulate the duration a taste receptor stays active would be usefulas a means of prolonging a desired taste or cutting off an unpleasantone. For a general review of GPCR signal transduction and methods ofassaying signal transduction, see, e.g., Methods in Enzymology, vols.237 and 238 (1994) and volume 96 (1983); Bourne, et al. (1991) Nature10:349:117-27; Bourne, et al. (1990) Nature 348:125-32; Pitcher, et al.(1998) Annu. Rev. Biochem. 67:653-92.

As indicated, ion flux assays can be carried out to determine whether acompound modulated GPR84 activity. Changes in ion flux can be assessedby determining changes in ionic polarization (i.e., electricalpotential) of the cell or membrane expressing a GPR84 protein. One meansto determine changes in cellular polarization is by measuring changes incurrent (thereby measuring changes in polarization) with voltage-clampand patch-clamp techniques (see e.g., Ackerman, et al. (1997) New Engl.J Med. 336:1575-1595). Whole cell currents are conveniently determinedusing a standard. Other known assays include: radiolabeled ion fluxassays and fluorescence assays using voltage-sensitive dyes (see, e.g.,Vestergarrd-Bogind, et al. (1988) J. Membrane Biol. 88:67-75; Gonzales &Tsien (1997) Chem. Biol. 4:269-277; Daniel, et al. (1991) J. Pharmacol.Meth. 25:185-193; Holevinsky, et al. (1994) J. Membrane Biology137:59-70). Generally, the compounds to be tested are present in therange from 1 pM to 100 mM.

The effects of the test compounds upon the function of GPR84 can bemeasured by examining any of the parameters described above. Anysuitable physiological change that affects GPCR activity can be used toassess the influence of a test compound on GPR84. When the functionalconsequences are determined using intact cells or animals, one can alsomeasure a variety of effects such as transmitter release, hormonerelease, transcriptional changes to both known and uncharacterizedgenetic markers (e.g., northern blots), changes in cell metabolism suchas cell growth or pH changes, and changes in intracellular secondmessengers such as Ca²⁺, IP₃, cGMP, or cAMP.

Preferred assays for GPCRs include cells that are loaded with ion orvoltage sensitive dyes to report receptor activity. Assays fordetermining activity of such receptors can also use known agonists andantagonists for other G protein-coupled receptors as negative orpositive controls to assess activity of tested compounds. In assays foridentifying modulatory compounds (e.g., agonists, antagonists), changesin the level of ions in the cytoplasm or membrane voltage will bemonitored using an ion sensitive or membrane voltage fluorescentindicator, respectively. Among the ion-sensitive indicators and voltageprobes that can be employed are those disclosed in the Molecular Probes1997 Catalog.

Other assays can involve determining the activity of receptors which,when activated, result in a change in the level of intracellular cyclicnucleotides, e.g., cAMP or cGMP, by activating or inhibiting enzymessuch as adenylate cyclase. There are cyclic nucleotide-gated ionchannels, e.g., rod photoreceptor cell channels and olfactory neuronchannels that are permeable to cations upon activation by binding ofcAMP or cGMP (see, e.g., Altenhofen, et al. (1991) Proc. Natl Acad. Sci.USA 88:9868-9872 and Dhallan, et al. (1990) Nature 347:184-187). Incases where activation of the receptor results in a decrease in cyclicnucleotide levels, it may be preferable to expose the cells to agentsthat increase intracellular cyclic nucleotide levels, e.g., forskolin,prior to adding a receptor-activating compound to the cells in theassay. Cells for this type of assay can be made by co-transfection of ahost cell with DNA encoding a cyclic nucleotide-crated ion channel, GPCRphosphatase and DNA encoding a receptor (e.g., certain glutamatereceptors, musearinic acetylcholine receptors, dopamine receptors,serotonin receptors, and the like), which, when activated, causes achange in cyclic nucleotide levels in the cytoplasm.

In addition, the uptake of fluorescently labeled fatty acids via CD36can be used as an indication of activity. Such an assay is commerciallyavailable from sources such as Molecular Devices (Sunnyvale, Calif.) foruse with FLIPR.

Non-human animals expressing GPR84, can also be used for receptorassays. Such expression can be used to determine whether a test compoundspecifically binds to a mammalian taste transmembrane receptorpolypeptide in vivo by contacting a non-human animal stably ortransiently transfected with a nucleic acid molecule of the inventionwith a test compound and determining whether the animal reacts to thetest compound by specifically binding to the receptor polypeptide.

Animals transfected or infected with the vectors of the invention areparticularly useful for assays to identify and characterizetastants/ligands that can bind to a specific or sets of receptors. Suchvector-infected animals expressing human chemosensory receptor sequencescan be used for in vivo screening of tastants and their effect on, e.g.,cell physiology (e.g., on taste neurons), on the CNS, or behavior.

Means to infect/express nucleic acids and vectors are well-known in theart. A variety of individual cell, organ, or whole animal parameters canbe measured by a variety of means. The nucleic acid molecule of theinvention can be for example expressed in animal taste tissues bydelivery with an infecting agent, e.g., adenovirus expression vector.

The compounds tested as modulators of GPR84 can be any small chemicalcompound, or a biological entity, such as a protein, nucleic acid orlipid. Typically, test compounds will be small chemical molecules andpeptides. Essentially any chemical compound can be used as a potentialmodulator or ligand in the assays of the invention, although most oftencompounds can be dissolved in aqueous or organic (especially DMSO-based)solutions are used. The assays are designed to screen large chemicallibraries by automating the assay steps and providing compounds from anyconvenient source to assays, which are typically run in parallel (e.g.,in microtiter formats on microtiter plates in robotic assays). It willbe appreciated that there are many suppliers of chemical compounds,including Sigma (St. Louis, Mo.), Aldrich (St. Louis, Mo.),Sigma-Aldrich (St. Louis, Mo.), Fluka Chemika-Biochemica Analytika(Buchs, Switzerland) and the like.

In one preferred embodiment, high throughput screening methods involveproviding a combinatorial chemical or peptide library containing a largenumber of potential therapeutic compounds (potential modulator or ligandcompounds). Such libraries are then screened in one or more assays, asdescribed herein, to identify those library members (particular chemicalspecies or subclasses) that display a desired characteristic activity.The compounds thus identified can serve as conventional “lead compounds”or can themselves be used as potential or actual therapeutics. GPR84modulators identified in accordance with the present invention can beused in any food product, confectionery, pharmaceutical composition, oringredient thereof to thereby modulate the taste of the product,composition, or ingredient in a desired manner.

1. An isolated nucleic acid molecule encoding CD36, G_(qi9), and Gprotein-coupled receptor 84 (GPR84) proteins.
 2. An expression vectorcomprising the nucleic acid molecule of claim
 1. 3. The expressionvector of claim 2, wherein expression of CD36, G_(qi9), and GPR84 isinducible.
 4. The expression vector of claim 2, wherein expression ofCD36, G_(qi)9, and GPR84 is constitutive.
 5. An isolated recombinanthost cell which co-expresses CD36, G_(qi9), and GPR84 proteins. 6-7.(canceled)